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, vaccination of calves with circulating maternal antibodies can elicit a cell-mediated immune response despite an apparent lack of a humoral response. 6,14 Bovine respiratory disease is associated with multiple pathogens including BRSV, BHV1, BVDV types 1

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in American Journal of Veterinary Research

Resistance to infection by respiratory disease viruses involves several aspects of the immune response. Serum virus-neutralizing antibodies are important and are the component of immunity that is most often measured as an indication of resistance

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in American Journal of Veterinary Research

Summary

The concentration of soluble fibrinogen derivatives (sfd) and protease and procoagulant activities were determined in cell-free supernatants of equine respiratory secretions obtained from horses with chronic pulmonary disease. The concentration of neutrophils was estimated from direct smears of the secretions. Lung specimens and smears of the secretions were evaluated for the presence of fibrin or fibrinogen by use of immunohistochemical methods.

Thirty-five of 80 specimens tested contained sfd. Respiratory secretions from horses with moderate or severe chronic pulmonary disease contained sfd more frequently than did secretions from mildly affected horses (P < 0.05). Respiratory secretions with vast numbers of neutrophils had significantly (P < 0.05) higher sfd concentrations than respiratory secretions with fewer neutrophils. Protease and procoagulant activities in respiratory secretion specimens were positively correlated with neutrophil content, clinical diagnosis, and sfd concentration.

Immunohistochemically, macrophages that stained for fibrin or fibrinogen were observed in direct smears of respiratory secretions from horses with moderate and severe chronic small airway disease, but not in smears from mildly affected horses. Fibrin or fibrinogen was detected in a few thickened alveolar septa from 10 horses with moderate or severe chronic small airway disease, but not in lungs from horses with mild or no evidence of chronic small airway disease. Fibrin or fibrinogen was detected in alveolar septa, granulomas, and on alveolar macrophages in lungs of all horses with chronic granulomatous and chronic bronchointerstitial pneumonia.

The presence of sfd in equine respiratory secretions may be an indicator of pulmonary inflammation.

Free access
in American Journal of Veterinary Research
in American Journal of Veterinary Research

Summary

Opsonized Rhodococcus equi activated the respiratory burst of resident alveolar macrophages (am) from adult horses in a logarithmic-linear, mass-related manner. The effect of R equi was not significantly different from that of equal masses of opsonized zymosan A. Therefore, R equi does not appear to attenuate the respiratory burst of equine am. The stimulatory effect of R equi was not reflected by increased production of superoxide anion (O2 -), but increased activity of the hexose monophosphate shunt was observed. These results suggest a similarity between the respiratory burst of am from horses and that of am from rabbits. We concluded that resident am from adult horses do not produce O2 - concurrently with an increase in activity of the hexose monophosphate shunt when stimulated with either opsonized zymosan A or opsonized R equi. This suggests that O2 - is not an important component of the antibacterial defenses of equine am. Whether equine am are incapable of producing O2 - or require different stimuli to produce it was not determined.

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in American Journal of Veterinary Research

Summary

Cell-mediated immunity was evaluated in intestinal, respiratory, and systemic lymphoid tissues of pigs exposed when 11 days old to virulent transmissible gastroenteritis virus (tgev), attenuated tgev, or porcine respiratory coronavirus (prcv), 3 antigenically related porcine coronaviruses with distinct enteric and respiratory tissue tropisms. Mononuclear cells were prepared from mesenteric lymph nodes (mln), bronchial lymph nodes (bln), and spleens of pigs and tested for virus-specific responses by use of lymphocyte proliferation assays. Vigorous mln and bln proliferation responses to virulent tgev and prcv, respectively, at postinoculation days 8 to 24 were strongly associated with prior detection of tgev in rectal swab samples and prcv in nasal swab samples. Gastrointestinal disease and intestinal virus replication, assessed on the basis of rectal virus shedding, were almost exclusively found in the virulent tgev-inoculated pigs, even though virulent tgev and a high dose of attenuated tgev elicited the highest proliferation responses in mln. Pigs exposed to prcv or attenuated tgev did not have clinical signs of disease, and only 1 pig given a high dose of attenuated tgev shed virus in feces. Porcine respiratory coronavirus replicated in the respiratory tract after either oronasal or aerosol inoculation of virus and induced strong bln, but not mln, proliferation responses. A high dose of attenuated tgev (4 × 108 plaque-forming units) was more effective than a lower dose of attenuated tgev (7 × 106 plaque-forming units) in eliciting significant lymphocyte proliferation in mln and bln. Cellular immune function, assessed on the basis of mitogen-induced proliferation of lymphocytes, was comparable for all 3 sources of lymphocytes and was not adversely affected by exposure to any of the 3 coronaviruses, nor did it vary with age of the pigs. The tissue tropism of tgev and prcv was associated with induction of virus-specific cell-mediated immune responses, as evidenced by substantial lymphocyte proliferation responses in mln and bln, mucosa-associated lymph nodes adjacent to the primary sites of virus replication. The failure of prcv strain ISU-1 to replicate in the intestinal tract correlated with poor virus-specific cellular immune responses in mln.

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in American Journal of Veterinary Research

Abstract

Objective

To determine whether the respiratory burst of neutrophils from bovine blood and milk can be analyzed by use of a fluorometric resazurin reduction assay.

Sample Population

Neutrophils were obtained from EDTA-anticoagulated blood of 7 dairy cows. Neutrophils also were isolated from milk samples of a cow intramammarily challenge exposed with Escherichia coli lipopolysaccharide.

Procedure

The respiratory burst of neutrophils was analyzed in parallel, using the conventional luminol-enhanced luminometric procedure and a novel fluorometric procedure with resazurin as the fluorogenic substrate. Opsonized zymosan and phorbol myristate acetate were used as stimulants. The mechanism of the fluorescent response was analyzed, using metabolic inhibitors to various cell functions. Luminometry and fluorometry were carried out in parallel, using microtitration tray-reading instruments.

Results

Stimulation of neutrophils induced resazurin reduction to resorufin and a fluorescent response. The luminescent response was transient, but the fluorescent response (build-up of fluorescent resorufin) was cumulative. Therefore, a single end-point measurement can be used for the fluorometric assay.

Conclusions

The proposed fluorometric microtitration tray technology is simple and has a high throughput capacity. The fluorometric and luminometric assays seem to have similar potential in the analysis of phagocyte functions. (Am J Vet Res 1997;58:601–607)

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in American Journal of Veterinary Research

SUMMARY

Newborn calves have a high susceptibility to bacterial infections, which may be related to the impaired neutrophil defense functions in newborns. The oxygen-dependent production of the free radical superoxide anion (O2 -) represents an important part of the leukocyte respiratory burst central to neutrophil-directed defenses against bacterial infection. Because protein kinase C (pkc) activation is considered to be an important step in the signal transduction pathway for the O2 - generating system, we compared O2 - production by newborn and adult bovine neutrophils stimulated with 3 different pkc agonists. When the phorbol ester phorbol 12-myristate 13-acetate (pma) was used, pkc-dependent O2 - generation from newborn neutrophils was significantly reduced (P < 0.01) for all concentrations of pma tested (10, 100, and 500 ng/ml). In addition, newborn neutrophils had a significantly (P < 0.01) reduced lag time for O2 - generation. Similar significantly (P < 0.01) reduced O2 - generation from newborn neutrophils was observed with an additional phorbol ester (phorbol 12, 13-dibutyrate); lag times were not calculated for phorbol 12, 13-dibutyrate. When O2 - generation was stimulated with a synthetic diacylglycerol analogue (1,2-dioctanoyl-sn-glycerol), less O2 - was generated from both adult and newborn neutrophils than was obtained with the phorbol esters, and newborn neutrophils produced significantly (P < 0.01) less O2 - only at 50 μM 1,2-dioctanoyl-sn-glycerol. To assess the importance of pkc and cyclic nucleotide-dependent protein kinases in the O2 --related signal-transduction pathways of bovine neutrophils, we determined the influence of a pkc inhibitor, H-7, and a cyclic nucleotide-dependent protein kinase inhibitor, HA-1004, on the respiratory burst of adult and newborn bovine neutrophils. Preincubation of neutrophils (15 minutes) with 100 or 500 μM H-7 inhibited subsequent pma-induced O2 - generation from newborn and adult neutrophils in a dose-dependent fashion, but preincubation with HA-1004 did not affect subsequent pma-induced O2 - generation from either newborn or adult neutrophils. The pkc inhibitor, H-7 (100 and 500 μM), induced a significantly (P < 0.01) prolonged lag time in adult, but not newborn, neutrophils; HA-1004 had no effect on lag time. These results indicate that pkc, but not cyclic nucleotide-dependent protein kinase, is important in the signal-transduction pathway leading to O2 - generation in appropriately stimulated adult and newborn bovine neutrophils, and that deficient respiratory burst activity in newborn neutrophils may be explained by altered activation of pkc.

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in American Journal of Veterinary Research

SUMMARY

A series of experiments was conducted to document tumor necrosis factor-α (tnf) activity in serum of swine after inoculation with Salmonella spp endotoxin and after oral or respiratory tract challenge exposure with live Salmonella spp. For experiment 1, a potentially lethal dose of S typhimurium endotoxin (25 μg/kg of body weight) was administered iv, and serum tnf activity was measured. High tnf (approx 700 IU/ml) activity at 1 to 2 hours after administration of the inoculum was associated with death, whereas lower tnf (approx 30 IU/ml) activity was associated with a general prolonged state of shock. For experiment 2, pigs were administered a nonlethal dose (5 μg/kg, iv) of either S typhimurium or S choleraesuis endotoxin. Difference in the ability to induce porcine serum tnf activity was not observed between strains. During experiment 3, pigs were inoculated with 104 colony-forming units of S typhimurium χ4232 either orally by gelatin capsule (gc) or by intranasal (in) instillation. A late serum tnf response (17 IU/ml) was measured at 6 weeks after in inoculation. A serum tnf response was not detected in gc-inoculated pigs. All tissues and feces were test-negative for S typhimurium prior to the 6-week tnf response. Serum tnf activity may be related to clearance of S typhimurium after respiratory tract exposure, but it is not important to or indicative of clearance of orally presented S typhimurium in swine. During experiment 4, pigs were inoculated with 106 colony-forming units of S typhimurium χ4232 similarly as for experiment 3. Challenge exposure with this medium-size dose of inoculum induced a prolonged peak serum tnf response (37 IU/ml) between 2 and 4 weeks after in inoculation. Again, serum tnf activity was not detected in gc-inoculated pigs. Data suggest that clearance of a medium-size dose (106) of inoculum may be influenced by the prolonged higher serum tnf activity. For experiments 5 and 6, pigs were inoculated in with 103, 106, 108, or 109 S choleraesuis χ3246. A measurable, yet statistically nonsignificant, serum tnf response was observed for all doses. Pigs inoculated by gc with 108 S choleraesuis χ3246 had similar results. High doses (> 106) of live S choleraesuis were associated with clinical signs of endotoxic shock. Clearance of S choleraesuis, or lack thereof, did not correlate with serum tnf activity.

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in American Journal of Veterinary Research

SUMMARY

To investigate the influence of humoral immunity on the severity of disease caused by infection with bovine respiratory syncytial virus (brsv), an experimentally induced infection study was performed on vaccinated and nonvaccinated calves. Fifteen weanling calves were allotted to 3 groups: 1 group of 6 calves was exposed to 2 live virus aerosols, 35 days apart; another group of 6 calves was vaccinated prior to the same aerosol exposures; and the remaining 3 calves served as controls. Clinical signs of infection were converted to a numerical score for evaluating disease severity. For 14 days after each virus exposure, brsv-specific IgG and IgM concentrations in serum and BRSV-specific IgA concentration in nasopharyngeal exudate and lung lavage fluid were measured by elisa. Serum brsv-specific IgG and IgM and secretory brsv- specific IgA concentrations did not correlate with disease sign expression. There was a strong correlation between viral isolation and disease scores. Vaccination prior to virus exposure appeared to have little or no effect on severity of the disease, but it did appear to affect disease persistence. Findings indicate that the immunoglobulins evaluated may be primarily protective in nature and do not contribute to disease severity.

Free access
in American Journal of Veterinary Research